1. Volume 117, Issue 5, Pages 1119-1127 (November 1999)
Expression of heregulin α, erbB2, and erbB3 and their influences on proliferation of gastric epithelial cells 
Hitoshi Noguchi, Choitsu Sakamoto, Ken Wada, Tomonori Akamatsu, Tohru Uchida, Atsushi Tatsuguchi, Hirofumi Matsui, Hirokazu Fukui, Takahiro Fujimori, Masato Kasuga 
Gastroenterology 
Volume 117, Issue 5, Pages (November 1999)
DOI: /S (99)
Copyright © 1999 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Immunoblot analysis of erbB receptor family in gastrointestinal cell lines. Cells were lysed, and the lysates were subjected to SDS-PAGE. The separated proteins were transferred onto nitrocellulose sheets, and blots were probed with (A) anti-EGFR antibodies, (B) anti-erbB2 antibody, (C) anti-erbB3 antibody, or (D) anti-erbB4 antibody. The lanes are as follows: 1, KATO-III; 2, JR-1; 3, MKN- 1; 4, MKN-28; 5, MKN-45; 6, MKN-74; 7, TMK-1; 8, IEC6; 9, RGM-1; 10, CHO cells; 11, CHO cells transfected with erbB4 cDNA.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

3. Fig. 2
PCR analysis of (A) erbB4 and (B) HRG-α RNA transcript expression in gastrointestinal cell lines. Total RNA was isolated, and RT-PCR was performed as described in Materials and Methods. (A) Lanes: 1, KATO-III; 2, JR-1; 3, MKN-1; 4, MKN-28; 5, MKN-45; 6, MKN-74; 7, TMK-1; 8, human gastric fibroblast; 9, HT-1080; 10, CHO cells; 11, CHO cells transfected with erbB4 cDNA. (B) Lanes: 1, KATO-III; 2, JR-1; 3, MKN-1; 4, MKN-28; 5, MKN-45; 6, MKN-74; 7, TMK-1; 8, human gastric fibroblast; 9, HT (C) β-Actin expression was determined by PCR using the same cDNA prepared in A and B. The lanes are the same as in B.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

4. Fig 3
Effects of HRG-α, TGF-α, and EGF on tyrosine phosphorylation of erbB receptors in MKN-28 cells. MKN-28 cells serum-starved for 24 hours were incubated with the indicated material for 10 minutes at 37°C. The cells were lysed and immunoprecipitated with antireceptor antibodies. Immunoprecipitates were first analyzed by immunoblotting with antiphosphotyrosine antibodies (PY). Thereafter, the nitrocellulose membrane was washed with 62.5 mmol/L Tris-HCl (pH6.7), 2% SDS, and 100 mmol/L 2-mercaptoethanol at 55°C for 1 hour and reprobed with each receptor antibody to confirm the same protein content in each lane.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

5. Fig 4
Ligand-induced heterodimerization of erbB receptor families. MKN-28 cells serum-starved for 24 hours were incubated with the indicated material for 10 minutes at 37°C. The cells were lysed and immunoprecipitated with antireceptor antibodies indicated by IP. Immunoprecipitates were analyzed by immunoblotting with antireceptor antibodies (Blot Ab).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

6. Fig 5
PI-3 kinase association with erbB3. MKN-28 cells were starved and incubated with the indicated material for 10 minutes at 37°C. The cells were lysed and immunoprecipitated with anti-erbB3 antibody. The immunoprecipitates were analyzed by immunoblotting with anti-p85 subunit antibody of PI-3 kinase.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

7. Fig 6
Immunohistochemical localization of (A) HRG-α and (B) erbB3 in the human gastric mucosa. (A) HRG-α immunoreactivity is present in scattered lamina propria mesenchymal cells corresponding to fibroblasts (arrow) in gastric mucosa. (B) ErbB3 immunoreactivity is present in glandular epithelial cells (arrow). (Original magnification 200× in A, 100× in B.)
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

8. Fig 7
Effect of coculture of MKN-28 cells and gastric fibroblasts on erbB3 tyrosine phosphorylation. Confluent coculture was lysed (lanes 2–4) or further cultured for an additional 24 hours under serum-free conditions and then lysed (lanes 5–7). Next, erbB3 receptor was immunoprecipitated with anti-erbB3 receptor antibody and subjected to Western blot analysis with antiphosphotyrosine antibody (Blot Ab: PY). The membrane was then washed and reprobed with anti-erbB3 antibody (Blot Ab: erbB3). Lane 1, MKN-28 cells stimulated with HRG-α; lanes 2 and 5, gastric fibroblasts alone; lanes 3 and 6, MKN-28 cells alone; lanes 4 and 7, fibroblasts and MKN-28 cells cocultured.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

9. Fig 8
Effects of HRG-α and TGF-α on DNA synthesis in (A) MKN-28 cells and (B) RGM-1 cells. These cells were cultured in the presence of various concentrations of HRG-α or TGF-α. Each data point represents the mean ± SEM of triplicate determinations from a representative of 3 experiments. *P < 0.05, **P < 0.01, significantly different from the respective control value.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

10. Fig 9
Effects of HRG-α and TGF-α on restitution of RGM-1 cells. Round wounds were established using an original device in confluent monolayers of RGM-1 cells. Wounded monolayers were cultured for 24 hours in 10 ng/mL rHRG-α or 10 ng/mL TGF-α. Photomicrographs were taken every 8 hours, and the uncovered area was measured. The vertical axis shows the residual uncovered area expressed as a percentage of the original wound area. Values represent the mean ± SEM of triplicate determinations from a representative of 3 experiments.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

11. Fig 10
Heterodimerization of EGFR family in MKN-28 cells. The horizontal black bar indicates the plasma membrane. HRG-α activates the erbB3/erbB2 and the erbB3/EGFR heterodimer in these cells. After dimer dissociation, phosphorylated erbB2 interacts with EGFR to form the erbB2/EGFR secondary heterodimer (broken arrow). Activated erbB3 is associated with the association of p85 subunit of PI-3 kinase (arrows). EGF and TGF-α induced only EGFR/erbB2 heterodimer formation. 1, EGFR; 2, erbB2; 3, erbB3; PI-3Kp85, p85 subunit of PI-3 kinase; P, phosphorylated tyrosine residues.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions